WO2022248917A1 - Procédé de détection et de quantification simultanées de listeria monocytogenes, salmonella spp, et escherichia coli produisant la toxine shiga (stec) - Google Patents

Procédé de détection et de quantification simultanées de listeria monocytogenes, salmonella spp, et escherichia coli produisant la toxine shiga (stec) Download PDF

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WO2022248917A1
WO2022248917A1 PCT/IB2021/054614 IB2021054614W WO2022248917A1 WO 2022248917 A1 WO2022248917 A1 WO 2022248917A1 IB 2021054614 W IB2021054614 W IB 2021054614W WO 2022248917 A1 WO2022248917 A1 WO 2022248917A1
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seq
polynucleotide
sequence
combinations
stec
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Inventor
Angélica REYES
Paola NAVARRETE
Gaétan CHINNICCI
Angel PARRA
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Universidad de Chile
Fundacion Copec - Universidad Catolica
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Universidad de Chile
Fundacion Copec - Universidad Catolica
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Priority to US18/563,954 priority Critical patent/US20250084490A1/en
Priority to BR112023024629A priority patent/BR112023024629A2/pt
Priority to EP21942863.8A priority patent/EP4347614A4/fr
Priority to PCT/IB2021/054614 priority patent/WO2022248917A1/fr
Priority to JP2023573155A priority patent/JP2024520068A/ja
Priority to CA3218648A priority patent/CA3218648A1/fr
Priority to KR1020237044554A priority patent/KR20240033215A/ko
Publication of WO2022248917A1 publication Critical patent/WO2022248917A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention refers to a method for simultaneous detection and quantification of Listeria monocytogenes, Salmonella spp. and Shiga toxin-producing Escherichia coli (STEC), from any kind of sample related with food production, including complex food matrices such as fish, meat, or fruit; or simple matrices such as water, or food contact surfaces.
  • STC Shiga toxin-producing Escherichia coli
  • Bacterial foodborne diseases produced by pathogenic bacteria or through their toxins are one of the main alimentary risks in food products, such as fruit, meat, or fish.
  • Shiga toxins provoke acute diarrhea, hemorrhagic colitis, and in some cases, acute kidney damage. Salmonella infections provoke vomiting, nausea and diarrhea. Listeriosis is a serious disease with symptoms ranging from febrile gastroenteritis to a severe invasive disease, and it especially affects pregnant women, children under 2 years, elders and immunocompromised people.
  • Bacterial culture is the traditional method to identify and quantify food borne pathogens in food. This method is based on identification of morphological, biochemical, physiological and immunological features from isolated bacterial colonies. However, even though this approach has been quite useful for many years, it has limitations such as being laborious and taking too long to obtain results. For example, the identification of any pathogen by culture method can take at least 4 days.
  • Time reduction for obtaining results thus becomes a critical aspect which directly impacts economics of agri-food industries. For example, it is commonly required to identify pathogens present in perishable food, so any delays in confirming food safety have important repercussions on storage costs while also reducing sales chances. Moreover, from a production process control perspective, rapid detection of possible contamination allows corrective actions to be taken on time, while also reducing possible cross contamination.
  • Rapidity has been accomplished through different molecular biology techniques, such as PCR, multiplex PCR and multiplex real-time PCR. Even though there are different PCR detection kits for these pathogens on the market, they are qualitative, in other words; they only screen for presence or absence of foodborne pathogens.
  • the quantification of microorganisms present on a matrix at industrial level is relevant in order to evaluate sanitizing and safety protocols in the production process.
  • it is still difficult to work with samples from food matrices using molecular biology tools due to their complexity and the presence of inhibitors from the food which may affect or interfere with the results, giving false negatives. This situation offers an opportunity for developing rapid, simultaneous and quantitative methods for foodborne pathogens, which is the application field of this invention.
  • Patent ES2540158 (Bl) refers to a simultaneous screening method based on multiplex PCR for seven different pathogens, including E. coli 0157:H7, L. monocytogenes and Salmonella spp. as targeted sequences.
  • An internal control corresponding to chimeric DNA is exclusively used in the amplification stage.
  • the present invention differs to this patent, in the PCR primers used, the internal control (not only used to evaluate the amplification stage) as well as in additional steps, which will be described later.
  • the inventors have solved this technical issue with a new method including a control bacterium, which is added to the food sample to be analyzed before separating microorganisms from the matrix, and allowing for a control of all steps of the process; thus, decreasing the number of false negative results and allowing quantitative results to be obtained.
  • FIG. 1 Calibration curves for the quantification of the pathogens: a) L monocytogenes, b) Salmonella spp. and c) STEC. All curves show Cq values versus each microorganism's concentration on samples (LoglO CFU/g or LoglO CFU/cm 2 ).
  • the invention refers to a method for simultaneous detection and quantification of Listeria monocytogenes, Salmonella spp. and Shiga toxin-producing Escherichia coli (STEC), from any kind of sample related to food production, including complex food matrices such as fish, meat, or fruit; or simple matrices such as water, or food contact surfaces.
  • the invention allows specific quantification of the above-mentioned pathogens simultaneously thanks to specificity of designed primers for the qPCR reaction. It is also highly reliable because the method includes a system to adequately control the quantification of pathogens within the matrix. This system comprises the inoculation of samples with a known concentration of a transformed microorganism (host) carrying a chimeric sequence, acting as an internal control host for the whole process.
  • host transformed microorganism
  • the invention consists in a method for simultaneous detection and quantification of Listeria monocytogenes, Salmonella spp. and Shiga toxin-producing Escherichia coli (STEC) from different matrices, including a host (like a bacterium) as internal control (I.C.) which ensures correct application of all process steps. It is important to note that the invention can be applied to detect and quantify 1 or simultaneously 2 or 3 of pathogens using the same internal control host. This invention then comprises the following steps:
  • the transformed bacterium species preferably used is an Escherichia coli, which does not produce Shiga toxins and is non-pathogenic.
  • the nature of the chimeric sequence also does not affect the method of detection and quantification of the pathogens, while the sequence included would be artificial and not- homologous to any bacterial genomes under study.
  • the inventors used a chimeric sequence constructed with a region with the sequence of one of the primers of the invention, separated by a region of 20 to 70 bp from a third region with the sequence of another of the primers of the invention. This way, the chimeric sequence incorporated in the control host carries a sequence of a forward primer selected from SEQ ID No.
  • the central linker region have 26 bp, and its sequence is that defined in SEQ ID No. 19.
  • said chimeric polynucleotide sequence is selected from the combinations of any of SEQ ID No 1, 3, 5 with any complementary of SEQ ID No 8, 10, 12, 14, 16, 18 (i.e. SEQ ID No39, 40, 41, 42, 43 and 44) , wherein the central-linker region is SEQ ID No 19, i.e. SEQ ID No 21, 45 to 61.
  • said chimeric polynucleotide sequence is selected from the combinations of any of SEQ ID No 7, 9, 11 with any complementary of SEQ ID No 2, 4, 6, 14, 16, 18 (i.e. SEQ ID No 36, 37, 38, 42, 43 and 44), wherein the central-linker region is SEQ ID No 19, i.e. SEQ ID No 62 to 79.
  • said chimeric polynucleotide sequence is selected from the combinations of any of SEQ ID No 13, 15, 17 with any complementary of SEQ ID No 2, 4, 6, 8, 10, 12 (i.e. SEQ ID No 36, 37, 38, 39, 40 and 41), wherein the central-linker region is SEQ ID No 19, i.e. SEQ ID No 20, 80 to 96.
  • SEQ ID No. 20 is formed by a region which is equivalent to Salmonella spp., the primer defined in SEQ ID No. 13, a 26 bp region sequence as defined in SEQ ID No. 19, and a region complementary to STEC, the primer defined in SEQ ID No. 8 (i.e. SEQ ID No 38).
  • SEQ ID No. 21 is formed by a region which is equivalent to Listeria monocytogenes, the primer defined in SEQ ID No. 1, a 26 bp region sequence as defined in SEQ ID No. 19, and a region complementary to STEC., the primer defined in SEQ ID No. 10 (i.e. SEQ ID No 40).
  • the method of invention further comprises a step to separate microorganisms out of the sample.
  • said step of microorganism separation is performed by filtration, centrifugation, sorting, magnetic beads or combinations thereof.
  • the method of invention further comprises a step of nucleic acids extraction from the sample.
  • nucleic acids detection is performed by PCR, RT-PCR, qPCR, digital PCR, LinDA, DNA microarray, PCR coupled with high-throughput sequencing, PCR DGGE/TTGE, or combinations thereof.
  • nucleic acids detection of L. monocytogenes is performed using primers comprising a sequence selected from SEQ ID No. 1 to 6, its derivatives, or combinations thereof; and when said nucleic acids detection of L. monocytogenes is performed by qPCR use probes comprising a sequence selected from SEQ ID No. 22 to 25, its derivatives, or combinations thereof.
  • nucleic acids detection of STEC is performed using primers comprising a sequence selected from SEQ ID No. 7 to 12, its derivatives, or combinations thereof; and when said nucleic acids detection of STEC is performed by qPCR, use probes comprising a sequence selected from SEQ ID No. 26 to 29, its derivatives, or combinations thereof.
  • nucleic acids detection of Salmonella spp. bacteria is performed using primers comprising a sequence selected from SEQ ID No. 13 to 18, its derivatives, or combinations thereof and when said nucleic acids of Salmonella spp. bacteria is performed by qPCR use probes comprising a sequence selected from SEQ ID No. 30 to 33, its derivatives, or combinations thereof.
  • nucleic acids detection of 1C is performed using as forward primer a sequence selected from SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, and 17, its derivatives, or combinations thereof; and as reverse primer a sequence selected from SEQ ID No 2, 4, 6, 8, 10, 12, 14, 16, and 18, its derivatives, or combinations thereof; and when said nucleic acids detection of 1C is performed by qPCR use probes comprising a sequence selected from SEQ ID No. 34 to 35, its derivatives, or combinations thereof.
  • said (c) step comprises a step of determining Listeria monocytogenes, Salmonella spp. bacteria and or Shiga toxin-producing Escherichia coli (STEC) concentration in a sample, interpolating the Cq value obtained by qPCR obtained in step (b) on a standard curve for each pathogen Listeria monocytogenes, Salmonella spp. bacteria and/or Shiga toxin producing Escherichia coli (STEC) concentration and validating it by the value for I.C. signal obtained in step (d).
  • the method of invention allows work with different types of samples, like biological sample selected from meat, poultry, seafood, sausages, dairy, fruits, vegetables, ready to eat foods, beverages, or water, or surfaces.
  • the I.C. host is added to the sample at a concentration of 10 2 -10 10 cells or CFU per mL
  • the polynucleotide to obtain an 1C useful to detect and quantify Listeria monocytogenes, Salmonella spp., bacteria and/or Shiga toxin-producing Escherichia coli (STEC) in a sample wherein said polynucleotide comprises: a sequence of a forward primer selected from SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15 and 17; a 26 bp central-linker region; and a complementary sequence to a reverse primer selected from SEQ ID No 2, 4, 6, 8, 10, 12, 14, 16 and 18 (i.e. SEQ ID No. 36 to 44).
  • said central-linker region in a preferred embodiment is SEQ ID No. 19.
  • the polynucleotide is selected from the combinations of any of SEQ ID No 1, 3, 5 with any complementary of SEQ ID No 8,10, 12, 14,16, 18 (i.e. SEQ ID No39, 40, 41, 42, 43 and 44), wherein the central-linker region is SEQ ID No 19, i.e. SEQ ID No 21, 45 to 61.
  • the polynucleotide is selected from the combinations of any of SEQ ID No 7, 9, 11 with any complementary of SEQ ID No 2, 4, 6, 14, 16, 18 (i.e. SEQ ID No 36, 37, 38, 42, 43 and 44), wherein the central-linker region is SEQ ID No 19, i.e. SEQ ID No.
  • the polynucleotide is selected from the combinations of any of SEQ ID No 13, 15, 17 with any complementary of SEQ ID No 2, 4, 6, 8, 10, 12 (i.e. SEQ ID No 36, 37, 38, 39, 40 and 41), wherein the central-linker region is SEQ ID No 19, i.e. SEQ ID No 20, 80 to 96.
  • said polynucleotide chimeric comprises a sequence selected from a sequence selected from SEQ ID No 20 (formed by SEQ ID No 13, 19 and 8) or SEQ ID No. 21 (formed by SEQ ID No 1, 19 and 10).
  • the polynucleotide can be included in plasmid, cassette, episome, DNA construct, or combinations thereof, and is carried by a host selected from bacteria, archaea or yeast. Wherein said host is, transformed, edited or transfected with said polynucleotide. In one preferred embodiment said host is Escherichia coii.
  • E. coii K12 (host) is used for the construction of the internal control, then transformed with vector pGEM ® -T Easy (Vector Systems- Promega Corporation) comprising a chimeric sequence as defined previously, and the transformants are selected using 100 pg/mL ampicillin.
  • vector pGEM ® -T Easy Vector Systems- Promega Corporation
  • the sample to be assessed is inoculated with a previously known quantity of the internal control host corresponding to a bacterium (£. coii K-12) transformed with a vector carrying chimeric sequence.
  • a 10-200 pL aliquot of a bacterial suspension of 10 2 -10 10 CFU/mL is added.
  • the internal control host is a bacterium transformed with the chimeric sequence, for the person skilled in the art it will be evident that there is a linear correlation between the concentration of the transformed bacteria and the chimeric sequence. So, depending on the stage of the process that is discussed, the term I.C. is used in this text to refer interchangeably to both terms, transformed control bacteria host or chimeric sequence.
  • the step referring to the microorganism's separation from the sample can be performed using any prior art methods depending on the nature of the sample.
  • separating all microorganisms from the sample is performed, for instance, from water only by filtration; from food by detachment washes using detergent solutions and then filtration; from surfaces by surface swabbing, resuspension in transport media and further filtration; and so on.
  • DNA extraction step proceeds, which may be accomplished using any techniques available in the prior art. Then, DNA is resuspended in nuclease-free water.
  • Quantitative PCR for L. monocytogenes, Salmonella spp., STEC and the chimeric sequence is performed using DNA extracted from the samples.
  • the qPCR result is validated in accordance with the Cq value for the chimeric sequence. In cases where the result is not consistent with the expected value, the measurement must be repeated.
  • Example 1 Quantification of Listeria monocytogenes, Salmonella spp. and Shiga toxin- producing Escherichia coli (STEC) in Salmon and a comparison with the traditional method.
  • target pathogens L. monocytogenes, Salmonella spp. and STEC were determined in parallel by using the method of the present invention as well as by the traditional "gold standard” culture method, applied to samples inoculated with different quantities of these pathogens.
  • the traditional method consists of seeding the sample in different culture plates containing specific media for each bacterium for 24 to 48 hours and counting the colony-forming units (CFU), then performing identification tests such as conventional PCR for each microorganism.
  • CFU colony-forming units
  • I.C. was obtained through transformation of E. coli K12 with vector pGEM ® -T Easy (Vector Systems - Promega Corporation) which carries a chimeric sequence defined as SEQ ID No. 31 designed with the reaction primers. To inoculate samples with I.C., this transformed bacterium was grown in LB (Luria-Bertani) broth plus 100 pg/mL ampicillin.
  • the Nitrocellulose filter containing the bacteria, was recovered using sterile tweezers and then put in a 2 mL centrifuge tube in order to proceed with DNA sample extraction.
  • bacteria DNA extraction was performed through standard extraction technology using phenobchloroform. This protocol allows lysing bacteria through enzymatic (lysozyme and proteinase) and chemical action (sodium dodecyl sulfate or SDS). Then the DNA is finally resuspended in nuclease-free water or a TE (Tris-EDTA) buffer. Once the DNA is obtained, qPCR is performed for detection of each pathogen and the chimeric sequence. For this 1 pL of resuspended DNA was used for each reaction, in triplicate.
  • Primers SEQ ID No. 1 and 2 were used for L. monocytogenes; primers SEQ ID No. 9 and 10 for STEC; primers SEQ ID No. 15 and 16 for Salmonella spp. and primers SEQ ID No. 1 and 10 for the chimeric sequence (I.C.).
  • the probes used for detection of L. monocytogenes SEQ ID No. 24), Salmonella spp. (SEQ ID No. 33), STEC (SEQ ID No. 26) and 1C (SEQ ID No. 34).
  • Table 1 for L. monocytogenes
  • Table 2 for Salmonella spp.
  • Table 3 for STEC.
  • the tables include the concentration of each microorganism added (Inoculum) to each sample, and the results obtained applying a Gold standard test.
  • tables show the Cq value obtained for the I.C. In order to validate the assay, the Cq value for the 1C must be in a 28 to 33.5 range.
  • Nl* non-inoculated. ** Detection limit of each method.
  • Nl* non-inoculated. ** Detection limit of each method.
  • Example 2 Quantification of Listeria monocytogenes, Salmonella spp. and Shiga toxin- producing Escherichia coli (STEC) in the transport media used for the surface testing system.
  • I.C. internal control host
  • the filter containing the bacteria, was recovered using sterile tweezers and then put in a 2 mL centrifuge tube.
  • DNA extraction of the bacteria present in the filter was performed through standard extraction technology using phenohchloroform, in accordance with the details of the previous example.
  • the DNA was resuspended in nuclease-free water or a TE (for Tris-EDTA) buffer.
  • Primers SEQ ID No. 3 and 4 were used for L. monocytogenes; primers SEQ ID No. 7 and 8 for STEC; primers SEQ ID No. 13 and 14 for Salmonella spp. and primers SEQ ID No. 13 and 8 for the chimeric sequence (I.C.).
  • the probes used for detection of L. monocytogenes SEQ ID No. 25), Salmonella spp. (SEQ ID No. 32), STEC (SEQ ID No. 27) and 1C (SEQ ID No. 35).
  • Table 4 results after using the method of the invention are shown in Table 4 for L. monocytogenes, in Table 5 for Salmonella spp. and in Table 6 for STEC.
  • the tables include the concentration of each microorganism added (Inoculum) to each sample, and the results obtained applying a Gold standard test.
  • the tables show the Cq value obtained for the I.C. in order to validate the assay, the Cq value for the 1C must be in a 28 to 33.5 range.
  • Nl* non-inoculated.
  • NA** gold standard quantification method is not available; *** Detection limit
  • Results show that it is possible to quantify L. monocytogenes, Salmonella spp., and STEC using the method of the invention at concentrations higher than Logio0.98, Logiol.16 and Logiol.04 CFU/cm 2 respectively, that corresponds to the detection limit of the method for this matrix.
  • values determined by the method of this invention are closer to the concentration inoculated to the surface sample, compared with those quantified by the gold standard test.
  • the method of the present invention quantifies three pathogens simultaneously while the gold standard processes each one separately.

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Abstract

L'invention concerne un procédé de détection et de quantification simultanées de Listeria monocytogenes, Salmonella spp. Et Escherichia Coli produisant la toxine Shiga (STEC), à partir de n'importe quel type d'échantillon associé à la production d'aliments, comprenant des matrices alimentaires complexes telles que des poissons, de la viande ou des fruits ; ou des matrices simples telles que l'eau, ou des surfaces de contact alimentaires. Le procédé selon l'invention permet une quantification spécifique des agents pathogènes mentionnés ci-dessus simultanément grâce à la spécificité d'amorces conçues pour la réaction qPCR. Il est également très fiable étant donné que le procédé comprend un système pour contrôler de manière adéquate la quantification d'agents pathogènes à l'intérieur de la matrice. Ce système comprend l'inoculation d'échantillons ayant une concentration connue d'un micro-organisme transformé (hôte) portant une séquence chimère, agissant en tant qu'hôte de commande interne pour l'ensemble du procédé.
PCT/IB2021/054614 2021-05-26 2021-05-26 Procédé de détection et de quantification simultanées de listeria monocytogenes, salmonella spp, et escherichia coli produisant la toxine shiga (stec) Ceased WO2022248917A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
US18/563,954 US20250084490A1 (en) 2021-05-26 2021-05-26 METHOD FOR SIMULTANEOUS DETECTION AND QUANTIFICATION OF Listeria monocytogenes, Salmonella spp., AND SHIGA TOXIN-PRODUCING Escherichia coli (STEC)
BR112023024629A BR112023024629A2 (pt) 2021-05-26 2021-05-26 Um método para detecção e quantificação simultânea ou independente de listeria monocytogenes, salmonella spp. e escherichia coli e polinucleotídeo
EP21942863.8A EP4347614A4 (fr) 2021-05-26 2021-05-26 Procédé de détection et de quantification simultanées de listeria monocytogenes, salmonella spp, et escherichia coli produisant la toxine shiga (stec)
PCT/IB2021/054614 WO2022248917A1 (fr) 2021-05-26 2021-05-26 Procédé de détection et de quantification simultanées de listeria monocytogenes, salmonella spp, et escherichia coli produisant la toxine shiga (stec)
JP2023573155A JP2024520068A (ja) 2021-05-26 2021-05-26 リステリア・モノサイトゲネス（Ｌｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓ）、サルモネラ属菌（Ｓａｌｍｏｎｅｌｌａ ｓｐｐ．）及び志賀毒素産生大腸菌（Ｓｈｉｇａ ｔｏｘｉｎ－ｐｒｏｄｕｃｉｎｇ Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）（ＳＴＥＣ）の同時検出及び定量化のための方法
CA3218648A CA3218648A1 (fr) 2021-05-26 2021-05-26 Procede de detection et de quantification simultanees de listeria monocytogenes, salmonella spp, et escherichia coli produisant la toxine shiga (stec)
KR1020237044554A KR20240033215A (ko) 2021-05-26 2021-05-26 리스테리아 모노사이토제네스, 살모넬라 속 및 시가 독소-생성 대장균(STEC)을 동시에 검출하고 정량화하는 방법(METHOD FOR SIMULTANEOUS DETECTION AND QUANTIFICATION OF Listeria monocytogenes, Salmonella spp., AND SHIGA TOXIN-PRODUCING Escherichia coli (STEC)

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PCT/IB2021/054614 WO2022248917A1 (fr) 2021-05-26 2021-05-26 Procédé de détection et de quantification simultanées de listeria monocytogenes, salmonella spp, et escherichia coli produisant la toxine shiga (stec)

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EP (1) EP4347614A4 (fr)
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BR (1) BR112023024629A2 (fr)
CA (1) CA3218648A1 (fr)
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Citations (8)

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